Interference of gadodiamide injection (OMNISCAN) on the colorimetric determination of serum calcium.

The interference of the non-ionic magnetic resonance contrast medium gadodiamide injection (OMNISCAN, Nycomed Imaging, Oslo, Norway) in the colorimetric determination of serum calcium has been investigated in commercial reconstituted serum, and in serum from rabbits and humans dosed with the contrast medium. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and ion-selective electrodes were used as reference methods for analysis of serum calcium. The results showed that the colorimetric reagent kit gave an apparent decrease in serum calcium after administration of a clinical dose of gadodiamide injection, and that the extent of interference is correlated to the concentration of the contrast medium. However, serum calcium was not changed when measured by means of an ion-selective electrode or ICP-AES. It is therefore recommended that colorimetric reagent kits should not be used for determination of serum calcium in samples taken within the first 24 h after administration of gadodiamide injection.

Complement activation and histamine release following administration of roentgen contrast media.

Anaphylactoid reactions following administration of reontgen contrast media (CM) have occasionally been described. In this investigation, blood samples for nonallergic human volunteers were exposed to the CM iodixanol (Visipaque), iohexol (Omnipaque), ioxaglate (Hexabrix) and metrizoate (Isopaque 350). The degree of activation of the complement cascade and the amount of free histamine in the samples were estimated. By using a hemolytic assay, a dose-independent complement consumption was detected when salt-free dilutions of the CM were added to human serum. Very little complement consumption was detectable when the concentrations, indicating that in the CM solutions were adjusted toward normal plasma concentrations, indicating that the lack of salts in the CM formulations was responsible for causing the consumption of complement rather than the CM molecules themselves. By using ELISA assay for determination of the terminal complement complex (TCC), no increase in TCC level was detected following the addition of iodixanol to human serum. The results indicate that iodixanol does not activate the complement cascade when added to human serum, and that it is unlikely that anaphylactoid reactions observed in man after CM administration are caused by CM-induced anaphylatoxins. No histamine release was observed following the addition of ioxaglate, metrizoate, iohexol or iodixanol to blood from nonallergic individuals.

Molecular cloning of a tissue-specific protein kinase (C gamma) from human testis--representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase.

Two different mammalian genes for the catalytic subunit (C) of cAMP-dependent protein kinase have previously been characterized (C alpha, C beta). In the present study, we report the molecular cloning of a third isoform of C, from a human testis cDNA library, as well as the isolation of human cDNAs for C alpha and C beta. This third form of C, which we will designate C gamma, is clearly derived from a distinct gene and shows a tissue-specific expression. A close evolutionary relation between C gamma and C alpha was suggested by nucleotide homologies (86% inside the open reading frame, 81% in the 3'-untranslated region). Thus, the C gamma cDNA cross-hybridized with the 2.8 kilobase (kb) C alpha mRNA, present at high levels in most human tissues, as well as with a 1.8 kb C gamma-specific mRNA, which was only found at detectable levels in human testis. However, at the amino acid level, C alpha and C beta showed a close relationship (93% homology), whereas C gamma diverged significantly from both C alpha (83%) and C beta (79%). Taken together with the tissue-specific expression of C gamma, this suggests a pressure on C gamma during evolution, acting to modulate it in a functionally specific way. Certain amino acid substitutions make C gamma a distinct member of the cAMP-dependent subfamily of protein kinases, and suggest that C gamma may be distinct in its protein substrate specificity or its interaction with the different regulatory subunits.

Biphasic regulation of the messenger ribonucleic acid coding for the estrogen receptor by cyclic adenosine 3':5'-monophosphate in tumor Leydig cells.

In this report we show that the mRNA level for the estrogen receptor (ER) is regulated by 8-bromo cyclic AMP (8-Br-cAMP) and human chorionic gonadotropin in a mouse tumor Leydig cell line (MA-10 cells). When the MA-10 cells were cultured in the presence of the cAMP analogue for varying time periods, a transient increase in the level of ER mRNA was observed. Short time incubation (0-2 h) with 8-Br-cAMP enhanced the expression of ER mRNA (2-fold), whereas longer times of incubation (6 h) had the opposite effect (the level of ER mRNA was reduced by 60-70%). The inhibitory effect of 8-Br-cAMP on ER mRNA was not counteracted by aminoglutethimide, an inhibitor of steroidogenic enzymes, indicating that this effect is not mediated via steroids (progesterone). Treatment of 8-Br-cAMP for 6 h caused a concentration-dependent inhibition of ER mRNA with a half-maximal effect of approximately 150 microM. Increasing concentrations of human chorionic gonadotropin for 6 h was also associated with a biphasic effect on the ER mRNA level. Low concentrations (0.20-0.40 ng/ml) increased ER mRNA in the MA-10 cells whereas the highest concentration (20 ng/ml) caused a suppression of this mRNA. In contrast to the biphasic effects observed for the ER mRNA, the level of the regulatory subunit type II beta of the cAMP-dependent protein kinase (protein kinase A) was enhanced in a concentration-dependent manner by human chorionic gonadotropin. Furthermore, 8-Br-cAMP stimulated the mRNA for regulatory subunit type II beta (10- to 20-fold) by all concentrations examined (50-1000 microM). The observations reported here indicate that the expression of ER mRNA is regulated both by endogenously formed and exogenously added cAMP and that there may exist regulatory loops between the steroid and the cAMP/protein kinase A systems.

Cellular localization and age-dependent changes in mRNA for cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat testis.

Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger RNA levels were studied by Northern analysis using available cDNA probes. The regulatory subunit (R) designated RII51 was found to be predominantly expressed in cAMP-stimulated Sertoli cells and tumor Leydig cells. Much lower levels were found in cultured peritubular cells and germ cells. A 2.9- and 3.2-kb mRNA for the RI subunit were found at about similar levels in all cell types, whereas the smaller 1.7-kb mRNA was expressed in high levels in germ cells. Also, the catalytic subunit (C) of cAMP-dependent protein kinase, designated C alpha, was expressed in all cell types; the highest mRNA levels for this subunit were found in germ cells and in tumor Leydig cells. The 1.7-kb mRNA for androgen-binding protein (ABP) was abundant in cAMP-stimulated Sertoli cells and was not present in other cell types of the testis. Furthermore, the cellular localization of the cAMP-dependent protein kinase subunits was also supported by developmental studies. The mRNA level of the RII51 3.2-kb species was relatively constant until Day 30, after which there was a tendency to decrease. A 1.6-kb message first appeared at greater ages. The mRNA for the smaller 1.7-kb species of RI, as well as the C alpha, showed a significant increase during development, supporting an enrichment of these mRNAs in germ cells. Messenger RNA levels for ABP were not detected in testis from 5- to 10-day-old rats but increased up to Day 30. After this age, mRNA for ABP revealed an age-dependent decrease, which parallels the relative increase of germ cells in the testis. In summary, these results demonstrate a clear pattern of cellular localization of the various mRNA species for subunits of the cAMP-dependent protein kinase in the rat testis.

"Differential displacement"--a simple method to study receptors for 1,25-dihydroxyvitamin D3 in the presence of contaminating serum vitamin D binding protein.

This report describes a simple procedure to quantitate receptors for 1,25-dihydroxyvitamin D3 in the presence of contaminating serum vitamin D binding protein. The method ("differential displacement") takes advantage of the greatly different rates of dissociation of 1,25-dihydroxyvitamin D3 from the serum vitamin D binding protein (t 1/2 less than 5 min) and from the receptor (t 1/2 greater than 120 h) at 0 degrees C. The principle of "differential displacement" can be used for other steroid receptors as well, and in combination with a variety of different binding assays, provided they are performed under conditions where the dissociation of the steroid from the receptor is slow.

Properties and compartmentalization of the testicular receptor for 1,25-dihydroxyvitamin D3.

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) with properties similar to 1,25-(OH)2D3 receptors in other tissues. The receptor sediments at 3.5 +/- 0.2 S20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH)2D3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH)2D3 (Kd = 3 X 10(-11) M), but low capacity (Nmax = 8 fmol/mg protein) and is specific for 1,25-(OH)2D3 (Affinity: 1,25-(OH)2D3 greater than 1,24(R),25-(OH)3D3 greater than 25-OH-D3 greater than 1 alpha-OH-D3 greater than 24(R),25-(OH)2D3 much greater than 17 beta-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [3H]1,25-(OH)2D3 and at 0 degrees C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature (t1/2 (0 degrees C) much greater than 48 h). At 0 degrees C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 X 10(7) M-1 min-1 and 2 X 10(-5) min-1, respectively. Receptors for 1,25-(OH)2D3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [3H]1,25-(OH)2D3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.

Glucagon-stimulated cyclic AMP production and formation of estradiol in Sertoli cell cultures from immature rats.

Addition of glucagon to the incubation medium of cultured Sertoli cells isolated from immature (19-day-old) rats resulted in a time- and concentration-dependent stimulation of cAMP accumulation measured both in the cells and in the medium. Maximal intracellular levels of cAMP were reached after 30 min, after which the levels decreased. In the medium cAMP levels reached a plateau after 6 h. The magnitude and kinetics of the responses were comparable to those observed with FSH in the same culture preparations. 1-Methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, greatly potentiated the magnitude of the effects of glucagon and FSH. Glucagon stimulated adenylate cyclase activity in isolated membrane preparations from similar cultures, and the concentration causing half-maximal stimulation (EC50) was approximately 300 ng/ml. Glucagon also stimulated aromatization in cultured Sertoli cells to the same extent as FSH. It is concluded that cultured Sertoli cells isolated from immature rats contain receptors for glucagon, coupled to adenylate cyclase, and that glucagon also stimulates aromatization of testosterone to estradiol.

Effects of FSH, isoproterenol, and cyclic AMP on the production of lactate and pyruvate by cultured Sertoli cells.

We have examined the hormonal regulation of the secretion of lactate and pyruvate from cultured rat Sertoli cells. FSH and isoproterenol caused 3-6-fold stimulation of lactate and pyruvate secretion, whereas ovine LH, TSH, and prolactin were ineffective. Dibutyryl cyclic AMP (10(-4) M) stimulated the secretion of lactate and pyruvate to the same extent as FSH. Much lower stimulation was observed when Sertoli cells from 43-day old rats were exposed to FSH or isoproterenol. FSH increased lactate secretion in a concentration-dependent manner. The concentration of FSH (NIH-S14) causing half-maximal stimulation of lactate secretion (150 ng/ml) was similar to that causing 50% maximal stimulation of Sertoli cell adenylyl cyclase. Both FSH and isoproterenol caused a time-dependent increase in lactate levels in the incubation medium during the first 6-9 hr after the addition of hormones, after which levels were constant or decreased. Thus, the production of lactate and pyruvate by cultured Sertoli cells is stimulated both by FSH and isoproterenol and these effects are exerted via cyclic AMP.